RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. RNAi targets include RNA from viruses and transposons (significant for some forms of innate immune response), and also plays a role in regulating development and genome maintenance. Small interfering RNA strands (siRNA) are key to the RNAi process, and have complementary nucleotide sequences to the targeted RNA strand. Specific RNAi pathway proteins are guided by the siRNA to the targeted messenger RNA (mRNA), where they “cleave” the target, breaking it down into smaller portions that can no longer be translated into protein. A type of RNA transcribed from the genome itself, microRNA (miRNA), works in the same way. RNAi occurs in both plants and animals, and is involved in many cellular functions, including regulating host gene expression and defense against infection by foreign organisms.
The process of RNAi begins by the presence of a double stranded RNA (dsRNA) in a cell, wherein the dsRNA contains a sense RNA having a sequence homologous to the target gene mRNA, and an antisense RNA having a sequence complementary to the sense RNA. The presence of dsRNA stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short inhibitory RNAs (siRNAs). Short inhibitory RNAs produced by Dicer are typically about 21 to about 23 nucleotides in length and contain about 19 base pair duplexes. siRNAs in turn stimulate an RNA-induced silencing complex (RISC) by incorporating one strand of siRNA into the RISC and directing the degradation of the homologous mRNA target.
Several methods have been used to deliver siRNAs to cells and suppress exogenous as well as endogenous gene expression. These methods include delivering synthetic siRNA molecules into cells, and vector-based methods in which siRNA is transcribed in a target cell by the vector. Certain vector-based siRNA delivery systems can result in persistent and effective suppression of gene expression, and, in certain cases expression of the siRNA can be used to select or kill cells containing the siRNA vector. Highly effective systems for delivering siRNA to a cell are established. In many vector-based methods, the siRNA is generated by the production of short hairpin RNA (shRNA), which contains a 19- to 29-bp RNA stem and a loop. In such a system, an RNA polymerase III promoter, such as H1 promoter and U6 promoter is used to drive transcription of shRNA. The shRNA is processed in the cell into siRNA through the action of the Dicer family of enzymes. Thus, the transcribed products mimic the synthetic siRNA duplexes and are as effective as the synthetic siRNA for suppressing their corresponding genes.
Certain embodiments described herein relate to a nucleic acid library for the production of shRNA in a mammalian cell.